Defining Cardiac Cell Populations and Relative Cellular Composition of the Early Fetal Human Heart

The human heart is primarily composed of cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Reliable identification of fetal cardiac cell types using protein markers is important for understanding cardiac development and delineating the cellular composition of the human heart during early development, which remains largely unknown. The aim of this study was to use immunohistochemistry (IHC), flow cytometry and RT-PCR analyses to investigate the expression and specificity of commonly used cardiac cell markers in the early human fetal heart (8-12 post-conception weeks). The expression of previously reported protein markers for the detection of cardiomyocytes (Myosin Heavy Chain (MHC) and Troponin I (cTnI)), fibroblasts (DDR2, Thy1, Vimentin), endothelial cells (CD31) and smooth muscle cells (α-SMA) were assessed. Flow cytometry revealed two distinct populations of cTnI expressing cells based on fluorescence intensity: cTnI High and cTnI Low . MHC positive cardiomyocytes were cTnI High , whereas MHC negative non-myocyte cells were cTnI Low . cTnI expression in non-myocytes was further confirmed by IHC and RT-PCR analyses, suggesting troponins are not cardiomyocyte-specific and may play distinct roles in non-muscle cells during early development. Vimentin was confirmed to be enriched in cultured fibroblast populations and flow cytometry revealed Vim High and Vim Low cell populations in the fetal heart. MHC positive cardiomyocytes were Vim Low whilst CD31 positive endothelial cells were Vim High . Based on the markers investigated, we estimate fetal human cardiomyocyte populations comprise 75-80% of total cardiac cells and exhibit the following marker profile: α-MHC + /cTnI High /Vim Low . For the non-cardiomyocyte population, we estimate they comprise 20-25% of total cardiac cells and exhibit the following marker profile: α-MHC - /cTnI Low /Vim High . Our study suggests the marker profiles and proportions of fetal cardiac populations are distinct from that of the adult heart.